The present invention relates to constructed bacterial strains that produce mosquito-toxic proteins. Compared with the highly mosquito-toxic strain B.t. israelensis, the constructed strains have improved characteristics, such as reduced spore number or increased variety of mosquito-toxic proteins. The invention also relates to a method of constructing improved mosquito-toxic strains of bacteria wherein the mosquito-toxin plasmid of B.t. israelensis is tagged with an antibiotic-resistance gene, thus permitting the detection of transfer of the mosquito-toxin B.t. israelensis plasmid from a donor bacteria strain into a recipient bacteria strain.
Bacillus thuringiensis (B.t.) is a gram-positive bacterium that produces proteinaceous crystalline inclusions during sporulation. These B.t. crystal proteins are often highly toxic to specific insects. Insecticidal activity has been identified for crystal proteins from various B.t. strains against insect larvae from the insect orders Lepidoptera (caterpillars), Diptera (mosquitos, flies) and Coleoptera (beetles).
Individual B.t. crystal proteins, also called delta-endotoxins or parasporal crystals or toxin proteins, can differ extensively in their structure and insecticidal activity. These insecticidal proteins are encoded by genes typically located on large plasmids, greater than 30 megadaltons (mDa) in size, that are found in B.t. strains. A number of these B.t. toxin genes have been cloned and the insecticidal crystal protein products characterized for their specific insecticidal properties. Hofte et al. provides a review of cloned B.t. toxin genes and crystal proteins (Microbiol. Rev., 1989, 53, 242-255).
The insecticidal properties of B.t. have long been recognized and B.t. strains have been incorporated into commercial biological insecticide products for over forty years. Commercial B.t. insecticide formulations typically contain dried B.t. fermentation cultures whose crystal protein is toxic to various insect species.
Traditional commercial B.t. bioinsecticide products are derived from xe2x80x9cwild-typexe2x80x9d B.t. strains, i.e., purified cultures of B.t. strains isolated from natural sources. Newer commercial B.t. bioinsecticide products are based on genetically altered B.t. strains, such as the transconjugant B.t. strains described in U.S. Pat. No. 5,080,897, issued Jan. 14, 1992, and U.S. Pat. No. 4,935,353, issued Jun. 19, 1990.
Various B.t. strains have been classified based on the reaction of the B.t. flagella with antibodies. A B.t. strain whose flagella react with a unique antibody is classified as a unique serovar, and over thirty different B.t. serovars or subspecies have been described (DeBarjac and Frachon, Entomophaga, 1990, 35, 233-240). Each B.t. subspecies often produces unique types of insecticidal crystal proteins. For example, B.t. subspecies kurstaki produces crystal proteins of approximatley 130 kilodaltons (kD) and 70 kD in size that are toxic to caterpillars, whereas B.t. subspecies tenebrionis produces a crystal protein of 72 kD which is toxic to beetles.
B.t. subspecies israelensis (B.t. israelensis) is a mosquito-toxic bacterium that produces at least four crystal proteins designated Cry4A, Cry4B, Cry4D and CytA that have been shown to be toxic to mosquito larvae. The B.t. crystal toxin gene designations have recently been revised (Crickmore et al., Microbiol. Molec. Biol. Rev., 1998, 62, 807-813). B.t. israelensis contains several native plasmids of approximate sizes 3.3, 4.2, 4.9, 10.6, 68, 75, 105 and 135 mDa (Gonzalez and Carlton, Plasmid, 1984, 11, 28-38). The genes for the Cry4A, Cry4B, Cry4D and CytA proteins are all carried on a single large plasmid, the mosquito-toxin plasmid, of approximately 75 mDa in B.t. israelensis (Gonzalez and Carlton, Plasmid, 1984, 11, 28-38). Gene cloning experiments have shown that recombinant non-B.t. israelensis bacteria containing cloned B.t. israelensis mosquito-toxin genes are generally less toxic to Aedes aegypti mosquitos than wild-type B.t. israelensis (Sekar and Carlton, Gene, 1985, 33, 151-158; Donovan et al., J. Bacteriol., 1988, 170, 4732-4738; Trisrisook et al., Appl. Envir. Microbiol., 1990, 56, 1710-1716; Bar et al., J. Invert. Pathology, 1991, 57, 149-158; Angsuthanasombat et al., FEMS Microbiol. Letts., 1992, 94, 63-68; Wu et al., Molec. Microbiol., 1994, 13, 965-972).
One explanation for the reduced mosquito toxicity seen with recombinant bacteria containing cloned B.t. israelensis mosquito-toxin-genes compared with wild-type B.t. israelensis is that all four B.t. israelensis crystal protein genes (i.e., cry4A, cry4B, cry4D and cytA) must be present in the same cell for maximum mosquito toxicity. Other factors of B.t. israelensis also contribute to its mosquito toxicity. For example, B.t. israelensis produces specific sugar residues that are attached to the B.t. israelensis crystal proteins (Pfannenstiel et al., J. Bacteriol., 1987, 169, 796-801) and these sugar residues make a significant contribution to the mosquito toxicity of B.t. israelensis (Muthukumar and Nickerson, App. Environ. Microbiol., 1987, 53, 2650-2655).
It has been found that certain strains of B.t. naturally transfer or conjugate their native plasmids to other strains of B.t. (Gonzalez and Carlton in xe2x80x9cGenetic Exchange,xe2x80x9d U. N. Streips, S. H. Goodgal, W. R. Guild and G. A. Wilson, Eds., 1982, p. 85-95, Marcel Dekker, Inc., New York). However, despite the ability of B.t. to naturally conjugate plasmids from one strain to another, the transfer of the 75 mDa mosquito-toxin plasmid from B.t. israelensis to a non-B.t. israelensis strain of B.t. has not been reported. Gonzalez and Carlton (Plasmid, 1984, 11, 28-38) have reported the lack of ability to transfer B.t. israelensis mosquito-toxin plasmid from B.t. israelensis to a non-israelensis strain of B.t. However, it is possible to transfer the mosquito-toxin plasmid from a donor B.t. israelensis strain to a recipient B.t. israelensis strain which had previously lost its 75 mDa mosquito-toxin plasmid.
Jensen et al. (Current Microbiol., 1996, 33, 228-236) have described an aggregation-mediated conjugation system of B.t. israelensis wherein a non-toxin plasmid of approximately 130 mDa, designated plasmid pXO16, was tagged with an antibiotic-resistance marker and the antibiotic-tagged, non-toxin plasmid was transferred into various recipient host cells. It should be emphasized that the antibiotic-tagged plasmid pXO16 described by Jensen et al. is known to transfer readily to non-B.t. israelensis strains. It is especially emphasized that the B.t. israelensis plasmid pXO16 does not carry mosquito-toxin genes. Although the non-toxin 130 mDa plasmid pXO16 of B.t. israelensis conjugates readily to non-B.t. israelensis strains, the mosquito-toxin 75 mDa plasmid of B.t. israelensis has not been found to conjugate into non-B.t. israelensis strains as shown by Gonzalez and Carlton (Plasmid, 1984, 11, 28-38).
The present invention relates to the 75 mDa mosquito-toxin plasmid of B.t. israelensis which is tagged with an antibiotic-resistance gene. The antibiotic-tagged plasmid is useful in detection of the rare event in which the mosquito-toxin plasmid transfers from a donor strain of B.t. into a recipient strain of B.t., the recipient strain being a non-B.t. israelensis strain.
The present invention also relates to constructed mosquito-toxic strains of B.t. which are non-B.t. israelensis strains and which contain the 75 mDa mosquito-toxin plasmid of B.t. israelensis. The mosquito-toxic strains are constructed by natural conjugation in which the antibiotic-tagged mosquito-toxin plasmid of B.t. israelensis is transferred from a donor strain into a non-israelensis recipient strain of B.t. It is within the scope of this invention to use other means, such as electroporation, to introduce the antibiotic-tagged, 75 mDa B.t. israelensis toxin-plasmid into recipient strains. Recipient strains have useful properties such as reduced spore numbers or improved ability to produce mosquito-toxic proteins compared with wild-type B.t. israelensis. 
The present invention relates to an antibiotic-tagged mosquito-toxin plasmid of approximately 75 mDa size of B.t. israelensis. The native B.t. israelensis mosquito-toxin plasmid of 75 mDa does not carry an antibiotic-tag and thus the present invention describes one method of adding an antibiotic-tag to the mosquito-toxin plasmid.
The present invention describes a novel method of overcoming the problem of detecting the very low rate of plasmid conjugation of the 75 mDa mosquito-toxin B.t. israelensis plasmid by providing a selectable antibiotic-resistance marker on the B.t. israelensis mosquito-toxin plasmid. The selectable antibiotic marker permits detection of the rare transfer of the B.t. israelensis mosquito-toxin plasmid from a donor cell into a non-israelensis recipient cell even when such a transfer occurs only once in a mixture of many thousands of donor and recipient cells.
The advantage of the present invention is that it permits the construction of non-B.t. israelensis strains of bacteria that have the complete 75 mDa mosquito-toxin plasmid of B.t. israelenesis and the constructed strains have equivalent mosquito toxicity as wild-type B.t. israelensis. In addition, the constructed strains have improved properties compared with B.t. israelensis as described in Examples 2, 3, 4 and 5.
According to the present invention mosquito-toxic strains of B.t. are constructed by the natural process of plasmid conjugation. The process of plasmid conjugation occurs when cells from two different strains of bacteria are combined and one or more plasmids are transferred from one of the bacteria strains, called the donor strain, to the second bacteria strain, called the recipient strain. Other means of introducing antibiotic-tagged plasmids into cells are well known in the art. For example, the technique of electroporation involves passing an electric current through a mixture of cells and antibiotic-tagged plasmids such that the membranes of the cell become permeable and a certain fraction of the cells receive the plasmid. The well known technique of introducing plasmids into cells by electroporation is described by Belliveau and Trevors (App. Environ. Microbiol., 1989, 55, 1649-1652).
It is within the scope of the present invention that the 75 mDa mosquito toxin plasmid of B.t. israelensis be tagged with an antibiotic marker by means known in the art other than the method described in Example 1. For example, the antibiotic tag could be attached to the mosquito-toxin plasmid by homologous DNA recombination. In the method of homologous recombination, a portion of DNA from 75 mDa mosquito-toxin plasmid is cloned into a xe2x80x9csuicidexe2x80x9d plasmid vector (i.e., a vector unable to replicate in B.t. but capable of replicating in another organism such as Escherichia coli). The vector contains an antibiotic resistance gene that becomes active when introduced into B.t. Introduction, by electroporation, of such a vector containing a portion of the mosquito-toxin plasmid DNA and a selectable antibiotic-resistance marker into B.t. israelensis, followed by selection for antibiotic resistance, results in integration, by homologous DNA recombination, of the vector plus antibiotic marker into the mosquito-toxin plasmid of B.t. israelensis, resulting in an antibiotic-tagged mosquito toxin plasmid.
Although the B.t. crystal toxin gene designations have recently been revised (Crickmore et al., Microbiol. Molec. Biol. Rev., 1998, 62, 807-813), for clarity, the former designations for B.t. israelensis crystal toxin will be used for discussion.